JouRNAL OF BACTOLOGY, Apr. 1981, p.

192-199
0021-9193/81/040192-08$02.00/0

VoL 146, No. 1

Ethanol Production by Thermophilic Bacteria: Metabolic

Control of End Product Formation in Thermoanaerobium
brockii
A. BEN-BASSAT, R. LAMED, AND J. G. ZEIKUS
Departnent of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received 16 July 1980/Accepted 2 February 1981

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process
rates, end product yields, and growth parameters. Fermentation of starch as
compared with glucose was associated with significant decreases in growth rate
and intracellular fructose-1,6-bisphosphate concentration and with a dramatic
increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation
in the presence of acetone was correlated with increased substrate consumption,
growth (both rate and yield), acetate yield, and quantitative reduction of acetone
to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol,
lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in
that H2, lactate, and ethanol levels decreased, whereas the acetate concentration
increased. Glucose fermentation in the presence of exogenous hydrogen was
associated with inhibition of endogenous H2 production and either increased
ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa)
of H2 or total growth inhibition at 1.0 atm (102 kPa). The effects of exogenous
hydrogen on glucose fermentation were totally reversed by the addition of
acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield,
and the formation of methane in lieu of monoculture reduced products. In
coculture, but not monoculture, T. brockii grew on ethanol as the energy source,
and acetate and methane were the end products as a direct consequence of
hydrogen consumption by the methanogen.

Biotechnological exploitation of anaerobic

bacteria for chemical and fuel production necessitates a knowledge of metabolic features that
control the rates, yields, and kinds of fermentation products formed by both pure and mixed
cultures. Regulation of end product formation in
pure cultures of anaerobic bacteria (i.e., intraspecies control) and mixed cultures (i.e., interspecies control) is poorly understood and appears complex, with multiple factors interacting
to control the rates and yields of specific product
formation. This includes parameters influencing
the flow of carbon and electrons in a given
metabolic path as well as species tolerance to
the fermentation products formed.
The parameters which govem the catabolic
electron flux in anaerobic bacteria are determined by thermodynamic considerations and
the specific activities of the catabolic enzymes as
a unit (7, 8, 10, 12). Thermophilic ethanologenic
bacteria examined to date (10, 19) employ a
branched pathway and form multiple fermentation products. Ethanol production is greatly in192

fluenced by regulation of electron flow at the

NAD+:ferredoxin oxidoreductase level, as exemplified in recent studies of Clostridium thermocellum and Thermoanaerobium brockii (10).
Fermentation of cellobiose by T. brockii yields
a reduced product ratio of 224 ethanol/20 H2/
352 lactate, whereas for C. thermocellum strain
LQRI, the ratio is 157 ethanol/286 H2/24 lactate.
In T. brockii, ethanol yield is higher as a consequence of electron flow from pyruvate to
ethanol via pyruvate:ferredoxin reductase, ferredoxin:NAD (and NADP) reductase, and
NADH (NADPH):acetaldehyde reductase. In C.
thermocellum strain LQRI, ferredoxin:NAD reductase and NADPH:acetaldehyde reductase
are not detectable during cellobiose fernentation. In C. thermocellum, H2 yield is higher
because of higher hydrogenase activity and the
absence of electron flow from reduced ferredoxin
or NADPH to lactate or ethanol. Higher lactate
yields in T. brockii corresponded with higher
intracellular levels of FDP, the allosteric activator of lactate dehydrogenase (10), and electron

VOL. 146, 1981

METABOLIC CONTROL OF ETHANOLOGENESIS

193

Metabolic measurements. All growth and metabolic experiments employed duplicate or more anaerobic culture tubes, and individual experiments were
duplicated or triplicated. Growth was determined by
measuring the increase in turbidity at 540 or 660 nm.
Optical density was quantified directly by insertion of
the anaerobic culture tubes into a Spectronic 20
(Bausch & Lomb, Inc., Rochester, N.Y.) spectrophotometer. Cell dry weight was determined by filtration
of the fermentation broth through a 0.45-gm membrane filter (Millipore Corp., Bedford, Mass.) filter
followed by drying overnight at 65C to a constant
weight. Glucose was determined with Statzyme reagent (Worthington Diagnostics, Chicago, Ill.). Fermentation products formed during growth were quantified by gas chromatography and by enzymatic analysis. Gases were analyzed, using the procedures described by Nelson and Zeikus (11). Organic alcohols
and acids were determined as described by Zeikus et
al. (21). L-Lactic acid was determined, using a standard
enzyme assay (1). Preparation and metabolic activity
analysis of cell extracts were as described by Lamed
'and Zeikus (9). Intracellular FDP concentration was
MATERIALS AND METHODS
Chemicals. All chemicals were reagent grade. En- determined as described previously (10).
zymes and coenzymes were obtained from Sigma
RESULTS
Chemical Co., St. Louis, Mo.; N2, H2, N2-C02 (95:5),
and H2-CO2 (80:20) were purchased from Matheson
of
fermentation
Control
by energy
Scientific, Inc., Joliet, Ill., and were passed through source(s). The amount of growth and specific
heated (310C) copper filings to remove traces of 02.
Organisms and cultivation procedures. T. fermentation product yields of T. brockii in combrockii neotype strain HTD4 (21) and Methanobac- plex medium varied with respect to the kind of
terium thermoautotrophicum strain YTB (20) were energy source(s) fermented (Table 1). Lactate
used in this study. Both strains were isolated from was the major fermentation product with gluthermal features in Yellowstone National Park and cose as the energy source, whereas ethanol was
were cultured as previously described (21, 22).
the major end product of starch fermentation.
Minimal medium (LPBB medium) contained (per The growth rate on starch (5-h doubling time)
liter of distilled water): NH4Cl, 1.0 g; MgCl2.6H20, 0.2 was approximately one-third of the rate on glug; KH2PO4, 0.30 g; Na2HPO4. 7H20, 2.0 g; trace mineral
solution, 10 ml; 2.5% FeSO4, 0.03 ml; 0.2% resazurin, 1 cose; starch fermentation was associated with
ml; and vitamin solution, 5 ml (21); 20 ml of 2.5% Na2S, lower intracellular FDP concentrations (4% of
glucose alone value) and a threefold-higher
was added after sterilization. The trace mineral solution contained (grams per liter of distilled water): ethanol/lactate ratio. Growth of T. brockii on
nitrilotriacetic acid neutralized to pH 6.5 with KOH, Trypticase (BBL Microbiology Systems)-yeast
12.8; FeSO4. 7H20, 0.1; MnCl2.4H20, 0.1; CoCl2- extract alone was slight, but it was significant in
6H20, 0.17; CaCl2.2H20, 0.1; ZnCl2, 0.1; CuCl2, 0.02; the presence of added acetone. Growth was also
H3BO3, 0.01; NaMoO4.2H20, 0.01; NaCl, 1.0; and significant (absorbance of 0.35 at 540 nm [Auo])

flow from pyruvate to NADH via ferredoxin:

NAD reductase.
General characterization of the catabolic enzyme activities of T. brockii suggested that electron flow from pyruvate to fermentation products was interconnected via several different oxidoreductase activities (9, 10). In addition, the
reversible NADP-linked alcohol dehydrogenase
of T. brockii was purified and shown to have a
broad substrate specificity that included acetone
and other ketones, aldehydes, and secondary
alcohols (R. Lamed and J. G. Zeikus, Biochem.
J., in press). Control of the metabolism of T.
brockii will be documented here by showing how
different energy sources and exogenous electron
donors and acceptors influence in vivo expression of catabolic enzyme activity (i.e., as measured by a change in fermentation products and
growth parameters).

Na2SeO3, 0.02. The culture medium gas phase contained N2. The pH of the medium was 7.2 after autoclaving. Minimal medium was supplemented with 0.1%
yeast extract (Difco Laboratories, Detroit, Mich.) and
0.5% glucose unless specified in the text. Complex
medium (TYEG) contained LPBB medium supplemented with 1% tryptone (Difco), 0.3% yeast extract,
and 0.5% glucose, which was autoclaved separately
before addition.
Most growth and metabolic studies were performed
in 24-mil anaerobic culture tubes (18 by 142 mm) from
Bellco (Bellco Glass, Inc., Vineland, N.J.) that contained 10 ml of medium and were sealed with no. 1
neoprene stoppers. Experiments that employed a H2
gas phase or methanogens were performed in anaerobic pressure tubes (18 by 142- mm) from Bellco that
contained 10 ml of medium and were sealed with a
pressure bung and a metal serum stopper crimp.

in acetone-miniimal medium supplemented with

0.6% yeast extract but not with Casamino Acids
or Trypticase soy broth alone. The specific substrates in yeast extract serving as an electron
donor(s) for energy metabolism under these conditions have not been identified. The addition of
acetone to glucose complex medium dramatically increased growth and acetate yield but
significantly decreased the intracellular FDP
concentration (40% of glucose alone value) and
the H2, lactate, and ethanol yields.
Control of fermentation by chemical electron acceptors and donors. Acetone was
shown to affect glucose metabolism of T. brockii
by functioning as a catabolic electron acceptor.
The addition of acetone to glucose miniimal me-

194

BEN-BASSAT, LAMED, AND ZEIKUS

dium significantly increased growth (both yield

and rate), glucose consumption, and acetate
yield and resulted in quantitative reduction of
acetone to isopropanol (Table 2; Fig. 1). The
effect of acetone on end product formation and
growth was concentration dependent, and increasing acetone concentrations correlated with
higher total end product formation and growth
yields. Acetone addition decreased the amount
of normal reduced end products (i.e., H2, ethanol,
and lactate) formed in favor of isopropanol production. With 200 mM acetone, hydrogen decreased more significantly than other reduced
products. The addition of acetone, regardless of
specific concentration, decreased the observed
product ratio of lactate formed/total products
formed. For example, this ratio was 0.58, 0.39,
and 0.18 during glucose fermentation alone or
with either 200 or 600 mM acetone, respectively.
In separate experiments, the addition of 30 to 50
mM 2-butanone or 2-methylcyclohexanone al-i
tered glucose metabolism of T. brockii in a manner similar to acetone, except these ketones were
reduced to their corresponding alcohols.
Table 3 compares the effect of acetone on

J. BACTERIOL.

pyruvate metabolism in growing cells and in cell

extracts. The effect of acetone on pyruvate-de-

pendent growth was the same as observed above

for glucose growth, except lactate was not a
significant end product. Acetone addition significantly decreased the amounts of H2, ethanol,
and lactate formed during growth and increased
the acetate yield. Likewise, acetone altered the
flow of electrons in an identical manner when
cell extracts decomposed pyruvate.
Experiments were initiated to examine
whether hydrogen, at a high partial pressure,
could reduce both endogenous and exogenous
electron acceptors during saccharide fermentation by T. brockii. Figure 1 and Table 4 show
the relation of H2 to glucose metabolism in the
presence and absence of acetone. Increasing the
partial pressure of H2 during glucose fermentation resulted in a decrease in the growth rate
and yield. Complete growth inhibition was
achieved at 1 atm (101 kPa) of hydrogen. Acetone addition reversed the growth inhibition
caused by H2 and increased the specific growth
rate. The effect of exogenous H2 on fermentation
product formation also displayed a pronounced

TABLE 1. Relation of energy sources to growth, [FDPJ, and fermentation product yields of T. brockii in
complex medium'
[FDP]
End products formed (total Amol per tube)
Ethanol/lacGrowth (AsAo) (nmol/mg
Addition
Ethano
t
Lactate
Acetate
Ethanol
of cells)
H2
2.0
16
11
33
8
0.15
None
0.70
162
15
110
11
52
1.3
Glucose
2.65
81
29
215
2
20
1.0
Starch
0.2
15
90
3
3
0.40
Acetone
0.75
20
262
15
4
21'
2.3
Glucose + acetone
a Culture tubes contained TYE medium and 0.5% glucose (250 ,umol) or starch (263 jumol of hexose) and 100
mM acetone (1 mmol) where indicated. The experiments were initiated with 0.2 ml of culture grown on TYEG
medium and incubated for 12 to 48 h at 650C.
TABLE 2. Effect of acetone on glucose metabolism by T. brockii in minimal mediuma
Acetone
Glucose
End products formed (total pmol per tube)
Addition
(ymol)

None
Acetone

(500)

Growth

(A,w0)

H2

Ethaol

Acetate

0.09
0.15

9
0

6
0

0
25

L-Lactate Isopropa9
4

nol
0
40

fermented
(total
pMol)

consumed
(total
pMol)
0
40

Carbon recovery

0.99
0
70
0
93
6
39
21
0.48
Glucose
1.17
94
98
94
128
71
30
9
0.72
Glucose +
acetone
(200)
1.0
269
123
269
92
139
6
16
0.85
Glucose +
acetone
(600)
a
Culture tubes contained LPBB medium, 0.1% yeast extract, and additions indicated. The glucose concentration was 0.5% (250 jAmol). The tubes were inoculated with 0.2 ml of a culture grown on TYEG medium and
incubated at 650C for 20 to 24 h. Carbon recovery represents the ratio of carbon in glucose consumed to carbon
in products formed. The amount of CO2 produced was estimated (i.e., 1 mol of C02 per mol of ethanol or
acetate).

METABOLIC CONTROL OF ETHANOLOGENESIS

VoL. 146, 1981

concentration dependence. An H2 concentration

of 0.3 to 0.5 atm (34 to 51 kPa) in glucose
fermentation most noticeably decreased net H2
formations and increased the ethanol/acetate
ratio, whereas at 1.0 atm of pressure, H2 production was not detectable, and all product yields
decreased dramatically. In the presence of acetone, hydrogen consumption occurred that was
dependent on the initial H2 partial pressure.
This effect correlated with increased isopropanol
formation at higher H2 partial pressures without
a noticeable change in final growth yield.
Control of fermentation by methanogens.
In nature and anaerobic digestors, the terminal
electron acceptors of microbial fermentative

195

processes are often methanogenic bacteria which

scavenge H2 and form CH4 as a consequence of

their energy-yielding metabolism. Recently, T.

brockii and M. thermoautotrophicum were
shown to coexist in the same niche as part of a
thermophilic microbial carbon cycle formed in
association with volcanic activity (20). Therefore, experiments were initiated to exmine the
effect of methanogenic activity on the metabolism of T. brockii. It is important to note that
M. thermoautotrophicum has a very limited
range (i.e., HrCO2 or CO) of energy sources for
growth (5, 22). Figure 2 demonstrates the effect
of M. thermoautotrophicum on glucose metabolism of T. brockii. In coculture, the dramatic
increase in growth rate and yield correlated with
increased acetate yield and decreased ethanol,
lactate, and hydrogen production. Methane was
2.0[
formed in lieu of the normal amounts of reduced
monoculture end products (i.e., H2, ethanol, and
1.0
lactate).
0 0.8
Most interesting, T. brockii was able to caIn) 0.6
tabolize substrates in the presence of M. thero
moautotrophicum that did not serve as energy
0.4
sources for growth in monoculture. Growth of
the coculture on ethanol involved the coupled
vr 0.21
metabolism of ethanol to acetate and H2 by T.
brockii and the conversion of H2-CO2 to CH4 by
M.
thermoautotrophicum (Table 5). The cocul0.1
ture also grew and converted yeast extract alone
to acetate and CH4. Cocultures did not utilize
4
12
0
20
16
lactic
acid as a carbon source for growth, nor did
TIME (HRS)
T. brockii monocultures grow on ethanol in the
FIG. 1. Effect ofH2 on T. brockii growth. Pressure presence of chemical electron acceptors, such as

49

tubes contained LPBB medium, 0.1% yeast extract, acetone.

0.5% glucose (OM Amol), and an N2 gasphase. Acetone
DISCUSSION
(700 pmol) or H2 (0.5 or 1 atm) or both were added as
The addition of exogenous electron donors or
indicated. The tubes were inoculated with 0.2 ml of
culture grown on TYEG medium.
acceptors or both (e.g., H2 or acetone) to T.
TABLE 3. Effect of acetone on pyruvate metabolism by T. brockii cultures and cell extracts
Growth
Cond
sGrwth
Conditions

Culturesa
Pyruvate
7h
24 h

Pyruvate
numd

Products formed (total jumol per tube)

Ethanol Acetate Lactate paol

(pmol)

H2

0.03
0.16

34
175

1
7

8
49

34
127

<0.5

0.16
0.36

81
251

<0.5
<0.5

<0.5

89
285

<0.5
<0.5

11

C2

Carbon
recovery

44
176

1.23
1.07

89
285

1.1

Pyruvate + acetone
7h
24 h

<0.5

85
267

1.13

Ceil extractsb
Pyruvate
ND
1
6
20
12
0
18
Pyruvate + acetone
ND
<0.1
1
29
1
26
26
a Culture tubes contained LPBB medium, 0.1% yeast extract, pyruvate (570 ,umol), and acetone (700
pmol) as
indicated. Tubes were inoculated at 65C and analyzed at 7 and 24 h as indicated.
b Enzyme reaction mixtures (1 ml) contained: 0.05 M potassium phosphate (pH 7.1), 400 ,umol of pyruvate, 0.1
,umol of coenzyme A, 5 mg of extract protein (0.2 ml), and 500 umol of acetone as indicated. The products were
measured after a reaction time of 1 min at 37C. ND, Not determined.

196

BEN-BASSAT, LAMED, AND ZEIKUS

J. BAcTERIoL.

TABLE 4. Effect of H2 on glucose fermentation products of T. brockua

H2 produced (+)
or consumed (-)

Addition

Total products per tube (ILmol)

(,mol)
Isopropanol
L-Lactate
Ethanol
Acetate
None
+44
235
166
38
0.3 atm of H2
+11
205
174
26
0.5atmofH2
ND
230
150
16
1.Oatm of H2
ND
50
20
19
Acetone
+6
495
112
23
316
0.3 atm of H2 + acetone
-77
508
135
21
300
0.5 atm of H2 + acetone
-118
552
116
20
287
1 atm of H2 + acetone
-153
615
164
18
198
a Pressure tubes contained LPBB medium, 0.1% yeast extract, 0.5% glucose (250 ,umol), and an N2 gas phase
(1 atm). Acetone (700 jAmol) or H2 (0.3 to 1 atm) or both were added as indicated. The tubes were inoculated
with 0.2 ml of cells grown on TYEG medium and incubated for 20 to 24 h at 650C. ND, Not detectable.
T.BROCKII

Co-CULTURE

1.6

160

E
140 a

1.41

/GROWTH

CD

1.21

120 F

1.01

100 a

0
0

U*.

O.8j

ACE TATE

/ /A

0.6

0.4
0.2

LOZ

01

AETOH

LACTATE

60
40

O ^ETON

2
1

GROWTH

80

,s,
a

0
0.
a

20

ACETATE

TI ME (HRS)

FIG. 2. Comparison of T. brockii glucose catabolism in mono- and coculture with M. thermoautotrophicum.
T. brockii monculbres were grown in pressure tubes that contained 5 ml of TYEG medium and an N2 gas
phase. Coculture experiments were performed as follows: the methanogen was grown to an optical density of
0.18 at 540 nm in pressure tubes that contained 5 ml of TYE medium and 3 atm (XX kPa) of H2-CO2 (80:20),
the gas phase was replaced with N2, and 0.5 ml of inoculum of T. brockii and glucose (125 pmol) was added.
All experimental tubes were incubated at 65C without shaking. ETOH, Ethanol.

brockii saccharide fermentations drastically altered metabolism by control of intraspecies electron flow. The in vivo physiological data presented here support the conclusions of in vitro
enzymological studies (10), in which the flow of
electrons from energy sources to end products of
T. brockii is mediated by interconnected oxidoreductases and common electron carriers [i.e.,
NAD(P) and ferredoxin]. These data (i.e.,
growth on ethanol) also suggest that the ferredoxin-NAD(P) oxidoreductase activities in T.
brockii are reversible ince the in vitro analysis
of enzymes only demonstrated electron flow
from ferredoxin to ethanol, lactate, and H2 (10).
Biochemical relationships that account for the
effect of acetone, hydrogen, and M. thermoau-

totrophicum on the metabolism of T. brockii are

shown in Fig. 3. The addition of hydrogen or
chemical and microbial electron acceptors influenced specific enzymatic reactions, and this altered carbon and electron flow. Acetone was
reduced by the NADP-linked alcohol dehydrogenase (reaction 5). Electron removal via acetone reduction to isopropanol decreased the
amount of normal reduced end products (i.e., H2,
ethanol, and lactate) and increased the metabolic rate and acetate yield. The acetone effect
was concentration dependent and could drastically decrease the yield of normal reduced products, probably as a consequence of the higher
affinity and activity of the organism's novel pyridine nucleotide-linked alcohol-aldehyde/ke-

VOL. 146, 1981

METABOLIC CONTROL OF ETHANOLOGENESIS

tone oxidoreductase towards acetone rather

than acetaldehyde (Lamed and Zeikus, in press).
In essence, acetone appears to alter the thermodynamics of electron flow during fermentation because of its recognition by the organism's
catabolic enzymes. Acetone, at a low concentration, decreased the amount of hydrogen more
significantly than it decreased that of lactate or
ethanol, probably as a consequence of more favorable thermodynamics associated with the
flow of electrons from reduced ferredoxin rather
than from NADH to isopropanol. In the presence of acetone, lactate was a less significant
product of pyruvate than glucose fermentation
because there is less NADH generation and
more thernodynamically favorable electron
flow.
Exogenous hydrogen was oxidized by hy-

197

drogenase (reaction 2), and increasing the H2

partial pressure led to complete inhibition of H2
production, altered reduced product yields via
reactions 4 to 9, and decreased metabolic rates.
Complete growth inhibition by 1 atm of H2 in
the absence but not in the presence of acetone
suggests that reduction of common electron carriers [i.e., NAD(P) and ferredoxin] by H2 is the
basis for H2 inhibition. The observed relationship between H2 oxidation and acetone reduction to isopropanol during glucose catabolism
demonstrated the reversibility of electron flow
from H2 to alcohol. Glucose fermentation in
coculture with M. thermoautotrophicum indicated that interspecies hydrogen metabolism
(reactions 2 and 3) altered electron flow, such
that H2 was formed by electrons generated from
reactions 10 and 9, in addition to altering pyru-

TABLE 5. Fermentation of ethanol by T. brockii and M. thermoautotrophicum

Growth

CUltUrea

Ethanol con-

Ciilti~(ODsu)b

(JAnol)

Products formed (total ,umol per tube)

Acetate
H2
CH4

T. brockii
0.07
0
10
9
0
M. thermoautotrophicum
0.01
0
0
0
0
T. brockii + M. thermoautotrophicum
0.3
187
200
1.6
167
T. brockii + M. thermoautotrophicum
0.1
20
1.8
55
(control; no ethanol)
a
Pressure tubes contained LPBB medium, 0.1% yeast extract, and 0.2% ethanol (435 ftmol/tube) as indicated.
The gas phase was C02-N2 (5:95) at 1 atm. The coculture experiments were inoculated with 1 ml of coculture
grown in the same medium. Monoculture experiments were inoculated with 0.2 ml of a T. brockii culture grown
on LPBB medium, 0.1% yeast extract, and glucose (0.5%; 250 ,mol) or with 0.5 ml of a M. thermoautotrophicum
culture grown on LPBB medium and 3 atm of H2-CO2 (80:20). All experimental tubes were incubated at 65C
for 48 h.
b ODseo, Optical density at 660 nm.
PYRUVATE

GLYCOLYSIS (GAP)
0

FERREDOX N 2 W METHANOGE N c H
9

C02

PY/

LACTATE

NADH A CoA
7
CH3CH
6

NADPH

/ ACETONE
ISOPROPANOL|

rETHANOL
FIG. 3. Metabolic control of catabolic electron flow by reversible oxidoreductases in T. brockii. The
numbers refer to the following enzyme activities: 1, pyruvate ferredoxin oxidoreductase; 2, hydrogenase; 3,
"methanogenases"; 4, ferredoxin-NADP oxidoreductase; 5, ethanol-NADP oxidoreductase; 6, ethanol-NAD
oxidoreductase; 7, NADH-acetyl coenzyme A oxidoreductase; 8, NADH-pyruvate reductase; 9, ferredoxinNAD oxidoreductase; and 10, glyceraldehyde-3-phosphate dehydrogenase. ACoA, Acetyl coenzyme A; GAP,
glyceraldehyde 3-phosphate. Note that the specific product of enzyme 5 depends on the substrate (i.e.,
acetaldehyde, ethanol, or acetone).

198

BEN-BASSAT, LAMED, AND ZEIKUS

vate-ferredoxin reductase activity (reaction 1).

Growth of the coculture on ethanol to H2 via
reactions 3, 4 to 7, and 9 occurred, provided that
reduced electron carriers were oxidized via
methanogen H2 consumption. This finding is
similar to that of studies of the "Methwnobacillas omelianskik' mixed culture (2, 16) which
concluded that growth of the "S-organism" on
ethanol was thermodynamically feasible only at
the low partidal pressures of H2 maintained by
the methanogen. Notwithstanding, the physiological properties and substrate range of T.
brockii are quite different from those of the Sorganism.
The data indicate that regulation of lactate
yield in T. brockii is complex and that it is
controlled by both electron flow and lactate
dehydrogenase activity. Lactate yield on starch
was lower than that on glucose as a consequence
of a lower [FDP] level caused by slower metabolic activity. On the other hand, lactate yield
was lower on glucose-acetone, probably as a
consequence of a lower [FDP] level caused by
increased metabolic activity and removal of pyruvate or NADH required for lactate dehydrogenase activity or both. The results discussed
above and the inability of T. brockii-M thermoautotrophicum cocultures to grow on lactate
support the irreversible character of the lactate
dehydrogenase of T. brockii (10) and the described regulatory properties for this enzyme in
lactic acid bacteria (16, 17).
Ultimately, the catabolic enzyme activities of
a bacterial species are probably determined by
the ecological niche in which the organism
evolved and by phylogenetic diversity. In this
regard, the metabolic interrelationships demonstrated for T. brockii and M. thermoautotrophicum are worth mentioning. These species are
found associated with anaerobic decomposition
of the algal-bacterial biomass formed in thermal
features (20). The data support some of the
suggested biological role(s) for methanogens in
nature (3, 6, 13, 16, 18). Namely, by regulation
of interspecies electron flow via H2 consumption,
methanogens can prevent formation of toxic
levels of intermediary metabolites (i.e., ethanol,
lactic acid, and hydrogen) and increase the rate
of organic matter decomposition. These data
clearly show that a methanogen can alter electron flow in fermentative bacteria that form
lactic acid in high yield, provided that the organisn also produces hydrogen via pyruvate dehydrogenase. Also worth mentioning is that
methanogen metabolismn also increases the substrate range of fermentative bacteria, probably
as a consequence of providing a thermodynamically favorable electron-accepting reaction.
Thus, in the presence of M. thermoautotrophi-

J. BACTERIOL.

cum, T. brockii fermented yeast extract and

ethanol.
Knowledge gained from an understanding of
catabolic electron flow in T. brockii may help
explain metabolic phenomena in different anaerobic bacterial species. Hrdependent growth inhibition of fermentative bacteria that form H2 is
a strain-specific character (10). For example,
Clostridium cellobioparum (4), Clostrdium
thernohydrosulfuricum (15), and T. brockii (10)
strains are inhibited but C. thermoceUlum (10,
14) and Clostridium pasteurianum (7, 8) strains
are not. This may be related to specific differences in the ferredoxin-NAD(P) oxidoreductase
activities, as demonstrated in T. brockii and C.
thermocelum strains (10). In this regard, we
observed the same general physiological findings
on electron control in C. thermohydrosulfuricum
strain 39E as those reported here for T. brockii.
The data also suggest that anaerobic bacterial
fermentations of readily soluble substrates (e.g.,
glucose) may be limited by the rate at which
reduced intracellular electron carriers (i.e.,
NADH, NADPH, and ferredoxin) are oxidized.
This speculation is based on the findings that
the metabolic rate of T. brockii was enhanced
by the addition of acetone or a methanogen and
was inhibited by hydrogen in the absence of an
exogenous electron acceptor.
These results may have practical significance
for commercial anaerobic bacterial fermentations. Manipulation of fermentation parameters
(e.g., energy source and exogenous electron donors-acceptors) that control intraspecies electron flow in ethanologens that form multiple
reduced end products can increase ethanol yields
in lieu of producing metabolic mutants that lack
hydrogenase or lactate dehydrogenase. In general, analysis of metabolic control of intra- and
interspecies electron flow in T. brockii fermentations suggests that ethanolic saccharide fermentations possess potentially higher product
yields (i.e., gram of product formed per gram of
substrate fermented) than do methanogenic fermentations but have lower process rates. The
major process limitations of bioethanol production with wild-type thermophilic bacterial
strains are low ethanol tolerance and final solvent concentrations of c1% in the fermentation
liquor. In methanogenic fermentations, end
product tolerance is not a problem, but control
of metabolic interactions between methanogenic
and non-methanogenic species is often process
limiting.
ACKNOWLEDGMENTS
This research was supported by the College of Agricultural
and Life Sciences, University of Wisconsin, Madison, and by
grant PFR 79-10084 from the National Science Foundation.

VOL. 146, 1981

METABOLIC CONTROL OF ETHANOLOGENESIS

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